Growth differentiation factor-8

ABSTRACT

A transgenic non-human animal of the species selected from the group consisting of avian, bovine, ovine and porcine having a transgene which results in disrupting the production of and/or activity of growth differentiation factor-8 (GDF-8) chromosomally integrated into the germ cells of the animal is disclosed. Also disclosed are methods for making such animals, and methods of treating animals with antibodies or antisense directed to GDF-8. The animals so treated are characterized by increased muscle tissue.

This application is a continuation-in-part application of U.S.application Ser. No. 08/525,596, filed Oct. 25, 1995, which is a 371application of PCT/US94/03019 filed on Mar. 18, 1994, which is acontinuation-in-part of U.S. application Ser. No. 08/033,923 filed onMar. 19, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to growth factors and specifically to anew member of the transforming growth factor beta (TGF-β) superfamily,which is denoted, growth differentiation factor-8 (GDF-8).

2. Description of Related Art

The Transforming growth factor β (TGF-β) superfamily encompasses a groupof structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer, et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325: 81-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs ((Weeks, et al.,Cell, 51: 861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Commun., 135: 957-964, 1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPs,osteogenin, OP-1) which can induce de novo cartilage and bone formation(Sampath, et al., J. Biol. Chem., 265:13198, 1990). The TGF-βs caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, hematopolesis, and epithelialcell differentiation (for review, see Massague, Cell 49:437, 1987).

The proteins of the TGF-β family are initially synthesized as a largeprecursor protein which subsequently undergoes proteolytic cleavage at acluster of basic residues approximately 110-140 amino acids from theC-terminus. The C-terminal regions, or mature regions, of the proteinsare all structurally related and the different family members can beclassified into distinct subgroups based on the extent of theirhomology. Although the homologies within particular subgroups range from70% to 90% amino acid sequence identity, the homologies betweensubgroups are signifi-cantly lower, generally ranging from only 20% to50%. In each case, the active species appears to be a disulfide-linkeddimer of C-terminal fragments. Studies have shown that when thepro-region of a member of the TGF-β family is coexpressed with a matureregion of another member of the TGF-β family, intracellular dimerizationand secretion of biologically active homodimers occur (Gray, A. et al.,Science, 247:1328, 1990). Additional studies by Hammonds, et al.,(Molec. Endocrin. 5:149, 1991) showed that the use of the BMP-2pro-region combined with the BMP-4 mature region led to dramaticallyimproved expression of mature BMP-4. For most of the family members thathave been studied, the homodimeric species has been found to bebiologically active, but for other family members, like the inhibins(Ling, et al., Nature, 321:779, 1986) and the TGF-Bs (Cheifetz, et al.,Cell, 48:409, 1987), heterodimers have also been detected, and theseappear to have different biological properties than the respectivehomodimers.

In addition it is desirable to produce livestock and game animals, suchas cows, sheep, pigs, chicken and turkey, fish which are relatively highin musculature and protein, and low in fat content.

Many drug and diet regimens exist which may help increase muscle andprotein content and lower undesirably high fat and/or cholesterollevels, but such treatment is generally administered after the fact, andis begun only after significant damage has occurred to the vasculature.Accordingly, it would be desirable to produce animals which aregenetically predisposed to having higher muscle content, without anyancillary increase in fat levels.

The food industry has put much effort into increasing the amount ofmuscle and protein in foodstuffs. This quest is relatively simple in themanufacture of synthetic foodstuffs, but has been met with limitedsuccess in the preparation of animal foodstuffs. Attempts have beenmade, for example, to lower cholesterol levels in beef and poultryproducts by including cholesterol-lowering drugs in animal feed (seee.g. Elkin and Rogler, J. Agric. Food Chem. 1990, 38, 1635-1641).However, there remains a need for more effective methods of increasingmuscle and reducing fat and cholesterol levels in animal food products.

SUMMARY OF THE INVENTION

The present invention provides a cell growth and differentiation factor,GDF-8, a polynucleotide sequence which encodes the factor, andantibodies which are immunoreactive with the factor. This factor appearsto relate to various cell proliferative disorders, especially thoseinvolving muscle, nerve, and adipose tissue.

In one embodiment, the invention provides a method for detecting a cellproliferative disorder of muscle, nerve, or fat origin and which isassociated with GDF-8. In another embodiment, the invention provides amethod for treating a cell proliferative disorder by suppressing orenhancing GDF-8 activity.

In another embodiment, the subject invention provides non-humantransgenic animals which are useful as a source of food products withhigh muscle and protein content, and reduced fat and choles-terolcontent. The animals have been altered chromosomally in their germ cellsand somatic cells so that the production of GDF-8 is produced in reducedamounts, or is completely disrupted, resulting in animals with decreasedlevels of GDF-8 in their system and higher than normal levels of muscletissue, preferably without increased fat and/or cholesterol levels.Accordingly, the present invention also includes food products providedby the animals. Such food products have increased nutritional valuebecause of the increase in muscle tissue. The transgenic non-humananimals of the invention include bovine, porcine, ovine and aviananimals, for example.

The subject invention also provides a method of producing animal foodproducts having increased muscle content. The method includes modifyingthe genetic makeup of the germ cells of a pronuclear embryo of theanimal, implanting the embryo into the oviduct of a pseudopregnantfemale thereby allowing the embryo to mature to full term progeny,testing the progeny for presence of the transgene to identifytransgene-positive progeny, cross-breeding transgene-positive progeny toobtain further transgene-positive progeny and processing the progeny toobtain foodstuff. The modification of the germ cell comprises alteringthe genetic composition so as to disrupt or reduce the expression of thenaturally occurring gene encoding for production of GDF-8 protein. In aparticular embodiment, the transgene comprises antisense polynucleotidesequences to the GDF-8 protein. Alternatively, the transgene maycomprise a non-functional sequence which replaces or intervenes in thenative GDF-8 gene.

The subject invention also provides a method of producing avian foodproducts having improved muscle content. The method includes modifyingthe genetic makeup of the germ cells of a pronuclear embryo of the aviananimal, implanting the embryo into the oviduct of a pseudopregnantfemale into an embryo of a chicken, culturing the embryo underconditions whereby progeny are hatched, testing the progeny for presenceof the genetic alteration to identify transgene-positive progeny,cross-breeding transgene-positive progeny and processing the progeny toobtain foodstuff.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a Northern blot showing expression of GDF-8 mRNA in adulttissues. The probe was a partial murine GDF-8 clone.

FIG. 1 b is a Southern blot showing GDF-8 genomic sequences identifiedin mouse, rat, human, monkey, rabbit, cow, pig, dog and chicken.

FIG. 2 shows nucleotide and predicted amino acid sequences of murineGDF-8 (FIG. 2 a), human GDF-8 (FIG. 2 b), rat GDF-8 (FIG. 2 c) andchicken GDF-8 (FIG. 2 d). The putative dibasic processing sites in themurine sequence are boxed.

FIG. 3 a shows the alignment of the C-terminal sequences of GDF-8 withother members of the TGF-β superfamily. The conserved cysteine residuesare boxed. Dashes denote gaps introduced in order to maximize alignment.

FIG. 3 b shows the alignment of the C-terminal sequences of GDF-8 fromhuman, murine, rat and chicken sequences.

FIG. 4 shows amino acid homologies among different members of the TGFsuperfamily. Numbers represent percent amino acid identities betweeneach pair calculated from the first conserved cysteine to theC-terminus. Boxes represent homologies among highly-related memberswithin particular subgroups.

FIG. 5 shows the sequence of GDF-8. Nucleotide and amino acid sequencesof murine (FIG. 5 a) (GenBank accession number U84005) and human (FIG. 5b) GDF-8 cDNA clones are shown. Numbers indicate nucleotide positionrelative to the 5′ end. Consensus N-linked g-lycosylation signals areshaded. The putative RXXR proteolytic cleavage sites are boxed.

FIG. 6 shows a hydropathicity profile of GDF-8. Average hydropho-bicityvalues for murine (FIG. 6 a) and human (FIG. 6 b) GDF-8 were calculatedusing the method of Kyte and Doolittle (J. Mol. Biol., 157: 105-132,1982). Positive numbers indicate increasing hydrophobicity.

FIG. 7 shows a comparison of murine and human GDF-8 amino acidsequences. The predicted murine sequence is shown in the top lines andthe predicted human sequence is shown in the bottom lines. Numbersindicate amino acid position relative to the N-terminus. Identitiesbetween the two sequences are denoted by a vertical line.

FIG. 8 shows the expression of GDF-8 in bacteria. BL21 (DE3) (pLysS)cells carrying a pRSET/GDF-8 expression plasmid were induced withisopropylthio-β-galactoside, and the GDF-8 fusion protein was purifiedby metal chelate chromatography. Lanes: total=total cell lysate;soluble=soluble protein fraction; insoluble=insoluble protein fraction(resuspended in 10 Mmn Tris pH 8.0, 50 mM sodium phosphate, 8 M urea,and 10 mM β-mercaptoethanol [buffer B]) loaded onto the column,pellet=insoluble protein fraction discarded before loading the column;flowthrough=proteins not bound by the column; washes=washes carried outin buffer B at the indicated pH's. Positions of molecular weightstandards are shown at the right. Arrow indicates the position of theGDF-8 fusion protein.

FIG. 9 shows the expression of GDF-8 in mammalian cells. Chinese hamsterovary cells were transfected with pMSXND/GDF-8 expression plasmids andselected in G418. Conditioned media from G418-resistant cells (preparedfrom cells transfected with constructs in which GDF-8 was cloned ineither the antisense or sense orientation) were concentrated,electrophoresed under reducing conditions, blotted, and probed withanti-GDF-8 antibodies and [¹²⁵I]iodoproteinA. Arrow indicates theposition of the processed GDF-8 protein.

FIG. 10 shows the expression of GDF-8 mRNA. Poly A-selected RNA (5 μgeach) prepared from adult tissues (FIG. 10 a) or placentas end embryos(FIG. 10 b) at the indicated days of gestation was electrophoresed onformaldehyde gels, blotted, and probed with full length murine GDF-8.

FIG. 11 shows chromosomal mapping of human GDF-8. DNA samples preparedfrom human/rodent somatic cell hybrid lines were subjected to PCR,electrophoresed on agarose gels, blotted, and probed. The humanchromosome contained in each of the hybrid cell lines is identified atthe top of each of the first 24 lanes (1-22, X, and Y). In the lanesdesignated M, CHO, and H, the starting DNA template was total genomicDNA from mouse, hamster, and human sources, respectively. In the lanemarked B1, no template DNA was used. Numbers at left indicate themobilities of DNA standards.

FIG. 12 a shows a map of the GDF-8 locus (top line) and targetingconstruct (second line). The black and stippled boxes represent codingsequences for the pro-and C-terminal regions, respectively. The whiteboxes represent 5′ and 3′ untranslated sequences. A probe derived fromthe region downstream of the 3′ homology fragment and upstream of themost distal HindIII site shown hybridizes to an 11.2 kb HindIII fragmentin the GDF-8 gene and a 10.4 kb fragment in an homologously targetedgene. Abbreviations: H, HindIII; X, Xba I.

FIG. 12 b shows a Southern blot analysis of offspring derived from amating of heterozygous mutant mice. The lanes are as follows: DNAprepared from wild type 129 SV/J mice (lane 1), targeted embryonic stemcells (lane 2), F1 heterozygous mice (lanes 3 and 4), and offspringderived from a mating of these mice (lanes 5-13).

FIG. 13 shows the muscle fiber size distribution in mutant and wild typelittermates. Smallest cross-sectional fiber widths were measured for (a)wild type (n=1761) and mutant (n=1052) tibialis cranial is or (b) wildtype (n=900) and mutant (n=900) gastrocnemius muscles, and fiber sizeswere plotted as a percent of total fiber number. Standard deviationswere 9 and 10 μm, respectively, for wild type and mutant tibialiscranialis and 11 and 9 μm, respectively, for wild type and mutantgastrocnemius muscles. Legend: o-o, wild type; _-_, mútant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a growth and differentiation factor,GDF-8 and a polynucleotide sequence encoding GDF-8. GDF-8 is expressedat highest levels in muscle and at lower levels in adipose tissue.

The animals contemplated for use in the practice of the subjectinvention are those animals generally regarded as useful for theprocessing of food stuffs, i.e. avian such as meat bred and egg layingchicken and turkey, ovine such as lamb, bovine such as beef cattle andmilk cows, piscine and porcine. For purposes of the subject invention,these animals are referred to as “transgenic” when such animal has had aheterologous DNA sequence, or one or more additional DNA sequencesnormally endogenous to the animal (collectively referred to herein as“transgenes”) chromosomally integrated into the germ cells of theanimal. The transgenic animal (including its progeny) will also have thetransgene fortuitously integrated into the chromosomes of somatic cells.

The TGF-β superfamily consists of multifunctional polypeptides thatcontrol proliferation, differentiation, and other functions in many celltypes. Many of the peptides have regulatory, both positive and negative,effects on other peptide growth factors. The structural homology betweenthe GDF-8 protein of this invention and the members of the TGF-β family,indicates that GDF-8 is a new member of the family of growth anddifferentiation factors. Based on the known activities of many of theother members, it can be expected that GDF-8 will also possessbiological activities that will make it useful as a diagnostic andtherapeutic reagent.

In particular, certain members of this superfamily have expressionpatterns or possess activities that relate to the function of thenervous system. For example, the inhibins and activins have been shownto be expressed in the brain (Meunier, et al., Proc. Natl. Acad. Sci.,USA, 85:247, 1988; Sawchenko, et al., Nature, 334:615, 1988), andactivin has been shown to be capable of functioning as a nerve cellsurvival molecule (Schubert, et al., Nature, 344:868, 1990). Anotherfamily member, namely, GDF-1, is nervous sys-tem-specific in itsexpression pattern (Lee, S. J., Proc. Natl. Acad. Sci., USA, 88:4250,1991), and certain other family members, such as Vgr-1 (Lyons, et al.,Proc. Natl. Acad. Sci., USA, 86:4554, 1989; Jones, et al., Development,111:531, 1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220,1992), and BMP-4 (Jones, et al., Development, 111:531, 1991), are alsoknown to be expressed in the nervous system. Because it is known thatskeletal muscle produces a factor or factors that promote the survivalof motor neurons (Brown, Trends Neurosci., 7:10, 1984), the expressionof GDF-8 in muscle suggests that one activity of GDF-8 may be as atrophic factor for neurons. In this regard, GDF-8 may have applicationsin the treatment of neurodegenerative diseases, such as amyotrophiclateral sclerosis or muscular dystrophy, or in maintaining cells ortissues in culture prior to transplantation.

GDF-8 may also have applications in treating disease processes involvingmuscle, such as in musculodegenerative diseases or in tissue repair dueto trauma. In this regard, many other members of the TGF-β family arealso important mediators of tissue repair. TGF-β3 has been shown to havemarked effects on the formation of collagen and to cause a strikingangiogenic response in the newborn mouse (Roberts, et al., Proc. Natl.Acad. Sci., USA 83:4167, 1986). TGF-β has also been shown to inhibit thedifferentiation of myoblasts in culture (Massague, et al., Proc. Natl.Acad. Sci., USA 83:8206, 1986). Moreover, because myoblast cells may beused as a vehicle for delivering genes to muscle for gene therapy, theproperties of GDF-8 could be exploited for maintaining cells prior totransplantation or for enhancing the efficiency of the fusion process.

The expression of GDF-8 in adipose tissue also raises the possibility ofapplications for GDF-8 in the treatment of obesity or of disordersrelated to abnormal proliferation of adipocytes. In this regard, TGF-βhas been shown to be a potent inhibitor of adipocyte differentiation invitro (Ignotz and Massague, Proc. Natl. Acad. Sci., USA 82:8530, 1985).

The term “substantially pure” as used herein refers to GDF-8 which issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. One skilled in the artcan purify GDF-8 using standard techniques for protein purification. Thesubstantially pure polypeptide will yield a single major band on anon-reducing polyacrylamide gel. The purity of the GDF-8 polypeptide canalso be determined by amino-.terminal amino acid sequence analysis.GDF-8 polypeptide includes functional fragments of the polypeptide, aslong as the activity of GDF-8 remains. Smaller peptides containing thebiological activity of GDF-8 are included in the invention.

The invention provides polynucleotides encoding the GDF-8 protein. Thesepolynucleotides include DNA, cDNA and RNA sequences which encode GDF-8.It is understood that all polynucleotides encoding all or a portion ofGDF-8 are also included herein, as long as they encode a polypeptidewith GDF-8 activity. Such polynucleotides include naturally occurring,synthetic, and intentionally manipu-lated polynucleotides. For example,GDF-8 polynucleotide may be subjected to site-directed mutagenesis. Thepolynucleotide sequence for GDF8 also includes antisense sequences. Thepolynucleotides of the invention include sequences that are degenerateas a result of the genetic code. There are 20 natural amino acids, mostof which are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included in the invention as long as the aminoacid sequence of GDF-8 polypeptide encoded by the nucleotide sequence isfunctionally unchanged.

Specifically disclosed-herein is a genomic DNA sequence containing aportion of the GDF-8 gene. The sequence contains an open reading framecorresponding to the predicted C-terminal region of the GDF-8 precursorprotein. The encoded polypeptide is predicted to contain two potentialproteolytic processing sites (KR and RR). Cleavage of the precursor atthe downstream site would generate a mature biologically activeC-terminal fragment of 109 and 103 amino acids for murine and humanspecies, respectively, with a predicted molecular weight ofapproximately 12,400. Also disclosed are full length murine and humanGDF-8 cDNA sequences. The murine pre-pro-GDF-8 protein is 376 aminoacids in length, which is encoded by a 2676 base pair nucleotidesequence, beginning at nucleotide 104 and extending to a TGA stop codonat nucleotide 1232. The human GDF-8 protein is 375 amino acids and isencoded by a 2743 base pair sequence, with the open reading framebeginning at nucleotide 59 and extending to nucleotide 1184. GDF-8 isalso capable of forming dimers, or heterodimers, with an expectedmolecular weight of approximately 23-30 KD (see Example 4). For example,GDF-8 may form heterodimers with other family members, such as GDF-11.

Also provided herein are the biologically active C-terminal fragments ofchicken (FIG. 2 c) and rat (FIG. 2 d) GDF-8. As shown in FIG. 3 b,alignment of the amino acid sequences of human, murine, rat and chickenGDF-8 indicate that the sequences are 100% identical in the C-terminalbiologically active fragment. Therefore, it would now be routine for oneof skill in the art to obtain the GDF-8 nucleic acid and amino acidsequence for GDF-8 from any species, including those provided herein, aswell as porcine, bovine, ovine, and piscine.

The C-terminal region of GDF-8 following the putative proteloyticprocessing site shows significant homology to the known members of theTGF-β superfamily. The GDF-8 sequence contains most of the residues thatare highly conserved in other family members and in other species (seeFIGS. 3 a and 3 b). Like the TGF-βs and inhibin βs, GDF-8 contains anextra pair of cysteine residues in addition to the 7 cysteines found invirtually all other family members. Among the known family members,GDF-8 is most homologous to Vgr-1 (45% sequence identity) (see FIG. 4).

Minor modifications of the recombinant GDF-8 primary amino acid sequencemay result in proteins which have substantially equivalent activity ascompared to the GDF-8 polypeptide described herein. Such modificationsmay be deliberate, as by site-directed mutagene-sis, or may bespontaneous. All of the polypeptides produced by these modifications areincluded herein as long as the biological activity of GDF-8 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biologi-cal activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-8 biological activity.

The nucleotide sequence encoding the GDF-8 polypeptide of the inventionincludes the disclosed sequence and conservative variations thereof. Theterm “conservative variation” as used herein denotes the replacement ofan amino acid residue by another, biologically similar residue. Examplesof conservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibod-ies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization techniques whichare well known in the art. These include, but are not limited to: 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences, 2) polymerase chain reaction (PCR) ongenomic DNA or cDNA using primers capable of annealing to the DNAsequence of interest, and 3) antibody screening of expression librariesto detect cloned DNA fragments with shared structural features.

Preferably the GDF-8 polynucleotide of the invention is derived from amammalian organism, and most preferably from mouse, rat, cow, pig, orhuman. GDF-8 polynucleotides from chicken, fish and other species arealso included herein. Screening procedures which rely on nucleic acidhybridization make it possible to isolate any gene sequence from anyorganism, provided the appropriate probe is available. Oligonucleotideprobes, which correspond to a part of the sequence encoding the proteinin question, can be synthesized chemically. This requires that short,oligopeptide stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction when the sequence isdegener-ate. This includes a heterogeneous mixture of denatureddou-ble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridiza-tion conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.9:879, 1981).

The development of specific DNA sequences encoding GDF-8 can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a doublestranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid-or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for GDF-8 peptides having at least one epitope, usingantibodies specific for GDF-8. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of GDF-8 cDNA.

DNA sequences encoding GDF-8 can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the GDF-8 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-8 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein 1, orpolyhedrin promoters).

Polynucleotide sequences encoding GDF-8 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. Preferably, themature C-terminal region of GDF-8 is expressed from a cDNA clonecontaining the entire coding sequence of GDF-8. Alternatively, theC-terminal portion of GDF-8 can be expressed as a fusion protein withthe pro-region of another member of the TGF-β family or co-expressedwith another pro-region (see for example, Hammonds, et al., Molec.Endocrin., 5:149, 1991; Gray, A., and Mason, A., Science, 247:1328,1990).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subse-quently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-8 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The invention includes antibodies immunoreactive with GDF-8 polypeptideor functional fragments thereof. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler, etal., Nature, 256:495, 1975). The term antibody as used in this inventionis meant to include intact molecules as well as fragments thereof, suchas Fab and F(ab′)₂′ Fv and SCA fragments which are capable of binding anepitopic determinant on GDF-8.

(1) An Fab fragment consists of a monovalent antigen-binding fragment ofan antibody molecule, and can be produced by digestion of a wholeantibody molecule with the enzyme papain, to yield a fragment consistingof an intact light chain and a portion of a heavy chain.

(2) An Fab′ fragment of an antibody molecule can be obtained by treatinga whole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

(3) An (Fab′)₂ fragment of an antibody can be obtained by treating awhole antibody molecule with the enzyme pepsin, without subse-quentreduction, A (Fab′)₂ fragment is a dimer of two Fab′ fragments, heldtogether by two disulfide bonds.

(4) An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

(5) A single chain antibody (“SCA”) is a genetically engineered singlechain molecule containing the variable region of a light chain and thevariable region of a heavy chain, linked by a suitable, flexiblepolypeptide linker.

As used in this invention, the term “epitope” refers to an antigenicdeterminant on an antigen, such as a GDF-8 polypeptide, to which theparatope of an antibody, such as an GDF-8-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteris-tics.

As is mentioned above, antigens that can be used in producingGDF-8-specific antibodies include GDF-8 polypeptides or GDF-8polypep-tide fragments. The polypeptide or peptide used to immunize ananimal can be obtained by standard recombinant, chemical synthetic, orpurification methods. As is well known in the art, in order to increaseimmunogenicity, an antigen can be conjugated to a carrier protein.Commonly used carriers include keyhole limpet hemocyanin (KLH),thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. Thecoupled peptide is then used to immunize the animal (e.g., a mouse, arat, or a rabbit). In addition to such carriers, well known adjuvantscan be administered with the antigen to facilitate induction of a strongimmune response.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e. cancer) develop as a result of a multistep process. TheGDF-8 polynucleotide that is an antisense molecule is useful in treatingmalignancies of the various organ systems, particularly, for example,cells in muscle or adipose tissue. Essentially, any disorder which isetiologically linked to altered expression of GDF-8 could be consideredsusceptible to treatment with a GDF-8 agent (e.g., a suppressing orenhancing agent). One such disorder is a malignant cell proliferativedisorder, for example.

The invention provides a method for detecting a cell proliferativedisorder of muscle or adipose tissue which comprises contacting ananti-GDF-8 antibody with a cell suspected of having a GDF-8 associateddisorder and detecting binding to the antibody. The antibody reactivewith GDF-8 is labeled with a compound which allows detection of bindingto GDF-8. For purposes of the inven-tion, an antibody specific for GDF-8polypeptide may be used to detect the level of GDF-8 in biologicalfluids and tissues. Any specimen containing a detectable amount ofantigen can be used. A preferred sample of this invention is muscletissue. The level of GDF-8 in the suspect cell can be compared with thelevel in a normal cell to determine whether the subject has aGDF-8-associated cell proliferative disorder. Preferably the subject ishuman.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibod-ies of the invention can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising the polypeptideof the invention. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and biolumi-nescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific antihapten antibod-ies.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled antibody is given a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled monoclonal antibody isadministered in sufficient quantity to enable detection of the sitehaving the antigen comprising a polypeptide of the invention for whichthe monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. Such dosages may vary, for example,depending on whether multiple injections are given, antigenic burden,and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radioisotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetra-acetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr and ²⁰¹Tl.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging or electron spin resonance (ESR). In general, anyconventional method for visualizing diagnostic imaging can be utilized.Usually gamma and positron emitting radioisotopes are used for cameraimaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The monoclonal antibodies of the invention can be used in vitro and invivo to monitor the course of amelioration of a GDF-8-associated diseasein a subject. Thus, for example, by measuring the increase or decreasein the number of cells expressing antigen comprising a polypeptide ofthe invention or changes in the concentration of such antigen present invarious body fluids, it would be possible to determine whether aparticular therapeutic regimen aimed at ameliorating theGDF-8-associated disease is effective. The term “ameliorate” denotes alessening of the detrimental effect of the GDF-8-associated disease inthe subject receiving therapy.

The present invention identifies a nucleotide sequence that can beexpressed in an altered manner as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Treatment includesadministration of a reagent which modulates activity. The term“modulate” envisions the suppression or expression of GDF-8 when it isover-expressed, or augmentation of GDF-8 expression when it isunderexpressed. When a muscle associated disorder is associated withGDF-8 overexpression, such suppressive reagents as antisense GDF-8polynucleotide sequence or GDF-8 binding antibody can be introduced intoa cell. In addition, an anti-idiotype antibody which binds to amonoclonal antibody which binds GDF-8 of the invention, or an epitopethereof, may also be used in the therapeutic method of the invention.Alternatively, when a cell proliferative disorder is associated withunderexpression or expression of a mutant GDF-8 polypeptide, a sensepolynucleotide sequence (the DNA coding strand) or GDF-8 polypeptide canbe introduced into the cell. Such muscle-associated disorders includecancer, muscular dystrophy, spinal cord injury, traumatic injury,congestive obstructive pulmonary disease (COPD), AIDS or cachecia.

Thus, where a cell-proliferative disorder is associated with theexpression of GDF-8, nucleic acid sequences that interfere with GDF-8expression at the translational level can be used. This approachutilizes, for example, antisense nucleic acid and ribozymes to blocktranslation of a specific GDF-8 mRNA, either by masking that mRNA withan antisense nucleic acid or by cleaving it with a ribozyme. Suchdisorders include neurodegenerative diseases, for example. In addition,dominant-negative GDF-8 mutants would be useful to actively interferewith function of “normal” GDF-8.

Antisense nucleic acids are DNA or RNA molecules that are comple-mentaryto at least a portion of a specific molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding forming a double-stranded molecule. Theantisense nucleic acids interfere with the translation of the mRNA sincethe cell will not translate a mRNA that is double-stranded.

Antisense of about 15 nucleotides are preferred, since they are easilysynthesized and are less likely to cause problems than larger moleculeswhen introduced into the target GDF-8—producing cell. The use ofantisense methods to inhibit the in vitro translation of genes is wellknown in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular Sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The present invention also provides gene therapy for the treatment ofcell proliferative or immunologic disorders which are mediated by GDF-8protein. Such therapy would achieve its therapeutic effect byintroduction of the GDF-8 antisense polynucleotide into cells having theproliferative disorder. Delivery of antisense GDF-8 polynucleotide canbe achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Especially preferred fortherapeutic delivery of antisense sequences is the use of targetedliposomes. In contrast, when it is desirable to enhance GDF-8production, a “sense” GDF-8 polynucleotide is introduced into theappropriate cell(s).

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV) Harvey murinesarcoma virus murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a GDF-8 sequence of interest into the viralvector, along with another gene which encodes the ligand for a receptoron a specific target cell, for example, the vector is now targetspecific. Retroviral vectors can be made target specific by attaching,for example, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome or attached to a viral envelope to allowtarget specific delivery of the retroviral vector containing the GDF-8antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsulation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for GDF-8 antisense polynucleo-tides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends BioChem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells in orderfor a liposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genes ofinterest at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Manning, etal., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phospha-tidylcompounds, such as phosphatidyiglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

Due to the expression of GDF-8 in muscle and adipose tissue, there are avariety of applications using the polypeptide, polynucleotide, andantibodies of the invention, related to these tissues. Such applicationsinclude treatment of cell proliferative disorders involving these andother tissues, such as neural tissue.

In addition, GDF-8 may be useful in various gene therapy proce-dures. Inembodiments where GDF-8 polypeptide is administered to a subject, thedosage range is about 0.1 ug/kg to 100 mg/kg; more preferably from about1 ug/kg to 75 mg/kg and most preferably from about 10 mg/kg to 50 mg/kg.

The data in Example 6 shows that the human GDF-8 gene is located onchromosome 2. By comparing the chromosomal location of GDF-8 with themap positions of various human disorders, it should be possible todetermine whether mutations in the GDF-8 gene are involved in theetiology of human diseases. For example, an autosomal recessive form ofjuvenile amyotrophic lateral sclerosis has been shown to map tochromosome 2 (Hentati, et al., Neurology, 42 [Supp 1.3]:201, 1992). Moreprecise mapping of GDF-8 and analysis of DNA from these patients mayindicate that GDF-8 is, in fact, the gene affected in this disease. Inaddition, GDF-8 is useful for distinguishing chromosome 2 from otherchromosomes.

Various methods to make the transgenic animals of the subject inventioncan be employed. Generally speaking, three such methods may be employed.In one such method, an embryo at the pronuclear stage (a “one cellembryo”) is harvested from a female and the transgene is microinjectedinto the embryo, in which case the transgene will be chromosomallyintegrated into both the germ cells and somatic cells of the resultingmature animal. In another such method, embryonic stem cells are isolatedand the transgene incorporated therein by electroporation, plasmidtransfection or microinjection, followed by reintroduction of the stemcells into the embryo where they colonize and contribute to the germline. Methods for microinjection of mammalian species is described inU.S. Pat. No. 4,873,191. In yet another such method, embryonic cells areinfected with a retrovirus containing the transgene whereby the germcells of the embryo have the transgene chromosomally integrated therein.When the animals to be made transgenic are avian, because avianfertilized ova generally go through cell division for the first twentyhours in the oviduct, microinjection into the pronucleus of thefertilized egg is problematic due to the inaccessibility of thepronucleus. Therefore, of the methods to make transgenic animalsdescribed generally above, retrovirus infection is preferred for avianspecies, for example as described in U.S. Pat. No. 5,162,215. Ifmicro-injection is to be used with avian species, however, a recentlypublished procedure by Love et al., (Biotechnology, 12, Jan. 1994) canbe utilized whereby the embryo is obtained from a sacrificed henapproximately two and one-half hours after the laying of the previouslaid egg, the transgene is microinjected into the cytoplasm of thegerminal disc and the embryo is cultured in a host shell until maturity.When the animals to be made transgenic are bovine or porcine,microinjection can be hampered by the opacity of the ova thereby makingthe nuclei difficult to identify by traditional differentialinterference-contrast microscopy. To overcome this problem, the ova canfirst be centrifuged to segregate the pronuclei for bettervisualization.

The “non-human animals” of the invention bovine, porcine, ovine andavian animals (e.g., cow, pig, sheep, chicken). The “transgenicnon-human animals” of the invention are produced by introducing“transgenes” into the gemmine of the non-human animal. Embryonal targetcells at various developmental stages can be used to introducetransgenes. Different methods are used depending on the stage ofdevelopment of the embryonal target cell. The zygote is the best targetfor micro-injection. The use of zygotes as a target for gene transferhas a major advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal., Proc. Natl. Acad. Sci. USA 82: 4438-4442, 1985). As a consequence,all cells of the transgenic non-human animal will carry the incorporatedtransgene. This will in general also be reflected in the efficienttransmis-sion of the transgene to offspring of the founder since 50% ofthe germ cells will harbor the transgene.

The term “transgenic” is used to describe an animal which includesexogenous genetic material within all of its cells. A “transgenic”animal can be produced by cross-breeding two chimeric animals whichinclude exogenous genetic material within cells used in reproduc-tion.Twenty-five percent of the resulting offspring will be transgenic i-e.,animals which include the exogenous genetic material within all of theircells in both alleles. 50% of the resulting animals will include theexogenous genetic material within one allele and 25% will include noexogenous genetic material.

In the microinjection method useful in the practice of the subjectinvention, the transgene is digested and purified free from any vectorDNA e.g. by gel electrophoresis. It is preferred that the transgeneinclude an operatively associated promoter which interacts with cellularproteins involved in transcription, ultimately resulting in constitutiveexpression. Promoters useful in this regard include those fromcytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,as well as those from the genes encoding metallothionin, skeletal actin,P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, andthymidine kinase. Promoters for viral long terminal repeats (LTRs) suchas Rous Sarcoma Virus can also be employed. When the animals to be madetransgenic are avian, preferred promoters include those for the chickenβ-globin gene, chicken lysozyme gene, and avian leukosis virus.Constructs useful in plasmid transfection of embryonic stem cells willemploy additional regulatory elements well known in the art such asenhancer elements to stimulate transcription, splice acceptors,termination and polyadenylation signals, and ribosome binding sites topermit translation.

Retroviral infection can also be used to introduce transgene into anon-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retro viral infection (Jaenich, R., Proc.Natl. Acad. Sci USA 73: 1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner, etal., Proc. Natl. Acad. Sci. USA 82: 6927-6931, 1985; Van der Putten, etal., Proc. Natl. Acad. Sci USA 82: 6148-6152, 1985). Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells (Van der Putten, supra; Stewart, etal., EMBO J. 6: 383-388, 1987). Alternatively, infection can beperformed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (D. Jahner et al., Nature 298: 623-628,1982) Most of the founders will be mosaic for the transgene sinceincorpora-tion occurs only in a subset of the cells which formed thetransgenic nonhuman animal. Further, the founder may contain variousretro viral insertions of the transgene at different positions in thegenome which generally will segregate in the offspring. In addition, itis also possible to introduce transgenes into the germ line, albeit withlow efficiency, by intrauterine retroviral infection of the midgestationembryo (D. Jahner et al., supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implant-ation embryoscultured in vitro and fused with embryos (M. J. Evans et al. Nature 292:154-156, 1981; M. O. Bradley et al., Nature 309: 255-258, 1984; Gossler,et al., Proc. Natl. Acad. Sci USA 83: 9065-9069, 1986; and Robertson etal., Nature 322: 445-448, 1986). Transgenes can be efficientlyintroduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter becombined with blastocysts from a nonhuman animal. The ES cellsthereafter colonize the embryo and contribute to the germ line of theresulting chimeric animal. (For review see Jaenisch, R., Science 240:1468-1474, 1988).

“Transformed” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant nucleic acid techniques, aheterologous nucleic acid molecule. “Heterologous” refers to a nucleicacid sequence that either originates from another species or is modifiedfrom either its original form or the form primarily expressed in thecell.

“Transgene” means any piece of DNA which is inserted by artifice into acell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode GDF-8, and include GDF-sense and antisensepolynucleotides, which may be expressed in a transgenic non-humananimal. The term “trans-genic” as used herein additionally includes anyorganism whose genome has been altered by in vitro manipulation of theearly embryo or fertilized egg or by any transgenic technology to inducea specific gene knockout. The term “gene knockout” as used herein,refers to the targeted disruption of a gene in vivo with complete lossof function that has been achieved by any transgenic technol-ogyfamiliar to those in the art. In one embodiment, transgenic animalshaving gene knockouts are those in which the target gene has beenrendered nonfunctional by an insertion-targeted to the gene to berendered non-functional by homologous recombination. As used herein, theterm “transgenic” includes any transgenic technology familiar to thosein the art which can produce an organism carrying an introducedtransgene or one in which an endogenous gene has been renderednon-functional or “knocked out.”

The transgene to be used in the practice of the subject invention is aDNA sequence comprising a modified GDF-8 coding sequence. In a preferredembodiment, the GDF-8 gene is disrupted by homologous targeting inembryonic stem cells. For example, the entire mature C-terminal regionof the GDF-8 gene may be deleted as described in the examples below.Optionally, the GDF-8 disruption or deletion may be accompanied byinsertion of or replacement with other DNA sequences, such as anon-functional GDF-8 sequence. In other embodiments, the transgenecomprises DNA antisense to the coding sequence for GDF-8. In anotherembodiment, the transgene comprises DNA encoding an antibody or receptorpeptide sequence which is able to bind to GDF-8. The DNA and peptidesequences of GDF-8 are known in the art, the sequences, localization andactivity disclosed in WO94/21681 and pending U.S. patent applicationSer. No. 08/033,923, filed on Mar. 19, 1993, incorporated by referencein its entirety. The disclosure of both of these applications are herebyincorpo-rated herein by reference. Where appropriate, DNA sequences thatencode proteins having GDF-8 activity but differ in nucleic acidsequence due to the degeneracy of the genetic code may also be usedherein, as may truncated forms, allelic variants and interspecieshomologues.

Therefore the invention also includes animals having heterozygousmutations in GDF-8. A heterozygote would likely have an intermedi-ateincrease in muscle mass as compared to the homozygote.

After an embryo has been microinjected, colonized with transfectedembryonic stem cells or infected with a retrovirus containing thetransgene (except for practice of the subject invention in avian specieswhich is addressed elsewhere herein) the embryo is implanted into theoviduct of a pseudopregnant female. The consequent progeny are testedfor incorporation of the transgene by Southern blot analysis of bloodsamples using transgene specific probes. PCR is particularly useful inthis regard. Positive progeny (GO) are crossbred to produce offspring(GI) which are analyzed for transgene expression by Northern blotanalysis of tissue samples. To be able to distinguish expression oflike-species transgenes from expression of the animals endogenous GDF-8gene(s), a marker gene fragment can be included in the construct in the3′ untranslated region of the transgene and the Northern probe designedto probe for the marker gene fragment. The serum levels of GDF-8 canalso be measured in the transgenic animal to establish appropriateexpression. Expression of the GDF-8 transgenes, thereby decreasing theGDF-8 in the tissue and serum levels of the transgenic animals andconsequently increasing the muscle tissue content results in thefoodstuffs from these animals (i.e. eggs, beef, pork, poultry meat,milk, etc.) having markedly increased muscle content, and preferablywithout increased, and more preferably, reduced levels of fat andcholesterol. By practice of the subject invention, a statisticallysignificant increase in muscle content, preferably at least a 2%increase in muscle content (e.g., in chickens), more preferably a 25%increase in muscle content as a percentage of body weight, morepreferably greater than 40% increase in muscle content in thesefoodstuffs can be obtained.

Thus, the present invention includes methods for increasing muscle massin domesticated animals, characterized by inactivation or deletion ofthe gene encoding growth and differentiation factor-8 (GDF-8). Thedomesticated animal is preferably selected from the group consisting ofovine, bovine, porcine, piscine and avian. The animal may be treatedwith an isolated polynucleotide sequence encoding growth anddifferentiation factor-8 which polynucleotide sequence is also from adomesticated animal selected from the group consisting of ovine, bovine,porcine, piscine and avian. The present invention includes methods forincreasing the muscle mass in domesticated animals characterized byadministering to a domesticated animal monoclonal antibodies directed tothe GDF-8 polypeptide. The antibody may be an anti-GDF-8, and may beeither a monoclonal antibody or a polyclonal antibody.

The invention includes methods comprising using an anti-GDF-8 monoclonalantibody as a therapeutic agent to inhibit the growth regulating actionsof GDF-8 on muscle cells. Muscle cells are defined to include fetal oradult muscle cells, as well as progenitor cells which are capable ofdifferentiation into muscle. The monoclonal antibody may be a humanized(e.g., either fully or a chimeric) monoclonal antibody, of any speciesorigin, such as murine, ovine, bovine, porcine or avian. Methods ofproducing antibody molecules with various combinations of “humanized”antibodies are well known in the art and include combining murinevariable regions with human constant regions (Cabily, et al. Proc. Natl.Acad. Sci. USA, =:3273, 1984), or by grafting the murine-antibodycomplementary determining regions (CDRs) onto the human framework(Richmann, et al., Nature 332:323, 1988). Other general references whichteach methods for creating humanized antibodies include Morrison, etal., Science, 229:1202, 1985; Jones, et al., Nature, 321:522, 1986;Monroe, et al., Nature 312:779, 1985; Oi, et al., BioTechniques, 4:214,1986; European Patent Application No. 302,620; and U.S. Pat. No.5,024,834. Therefore, by humanizing the monoclonal antibodies of theinvention for in vivo use, an immune response to the antibodies would begreatly reduced.

The monoclonal antibody, GDF-8 polypeptide, or GDF-8 polynucleotide (all“GDF-8 agents”) may have the effect of increasing the development ofskeletal muscles. In preferred embodiments of the claimed methods, theGDF-8 monoclonal antibody, polypeptide, or polynucleotide isadministered to a patient suffering from a disorder selected from thegroup consisting of muscle wasting disease, neuromuscular disorder,muscle atrophy or aging. The GDF-8 agent may also be administered to apatient suffering from a disorder selected from the group consisting ofmuscular dystrophy, spinal cord injury, traumatic injury, congestiveobstructive pulmonary disease (COPD), AIDS or cachechia. In a preferredembodiment, the GDF-8 agent is administered to a patient with musclewasting disease or disorder by intravenous, intramuscular orsubcutaneous injection; preferably, a monoclonal antibody isadministered within a dose range between about 0.1 mg/kg to about 100mg/kg; more preferably between about 1 ug/kg to 75 mg/kg; mostpreferably from about 10 mg/kg to 50 mg/kg. The antibody may beadministered, for example, by bolus injunction or by slow infusion. Slowinfusion over a period of 30 minutes to 2 hours is preferred. The GDF-8agent may be formulated in a formulation suitable for administration toa patient. Such formulations are known in the art.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the GDF-8protein, e.g. amount of tissue desired to be formed, the site of tissuedamage, the condition of the damaged tissue, the size of a wound, typeof damaged tissue, the patient's age, sex, and diet, the severity of anyinfection, time of administration and other clinical factors. The dosagemay vary with the type of matrix used in the reconstitution and thetypes of agent, such as anti-GDF-8 antibodies, to be used in thecomposition. Generally, systemic or injectable administration, such asintravenous (IV), intramuscular (IM) or subcutaneous (Sub-Q) injection.Administration will generally be initiated at a dose which is minimallyeffective, and the dose will be increased over a preselected time courseuntil a positive effect is observed. Subsequently, incremental increasesin dosage will be made limiting such incremental increases to suchlevels that produce a corresponding increase in effect, while takinginto account any adverse affects that may appear. The addition of otherknown growth factors, such as IGF I (insulin like growth factor I),human, bovine, or chicken growth hormone which may aid in increasingmuscle mass, to the final composition, may also affect the dosage. Inthe embodiment where an anti-GDF-8 antibody is administered, theanti-GDF-8 antibody is generally administered within a dose range ofabout 0.1 ug/kg to about 100 mg/kg.; more preferably between about 10mg/kg to 50 mg/kg.

Progress can be monitored by periodic assessment of tissue growth and/orrepair. The progress can be monitored, for example, x-rays,histomorphometric determinations and tetracycline labeling.

All references cited herein are hereby incorporated by reference intheir entirety.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alterna-tively be used.

EXAMPLE 1

To identify a new member of the TGF-β superfamily, degenerateoligonucleotides were designed which corresponded to two conservedregions among the known family members: one region spanning the twotryptophan residues conserved in all family members except MIS and theother region spanning the invariant cysteine residues near theC-terminus. These primers were used for polymerase chain reactions onmuse genomic DNA followed by subcloning the PCR products usingrestriction sites placed at the 5′ ends of the primers, pickingindividual E. coli colonies carrying these subcloned inserts, and usinga combination of random sequencing and hybridization analysis toeliminate known members of the superfamily. GDF-8 was identified from amixture of PCR products obtained with the primers SJL141:5′-CCGGAATTCGGITGG (G/C/A) A (G/A/T/C) (A/G) A (T/C) TGG (A/G) TI (A/G)TI (T/G) CICC-3′ (SEQ ID NO:1) SJL147: 5′-CCGGAATTC (G/A) CAI (G/C) C(G/A) CA (G/A) CT (GIA/T/C) TCIACI (G/A) (T/C) CAT-3′ (SEQ ID NO:2)

PCR using these primers was carried out with 2 μg mouse genomic DNA at94° C. for 1 min, 50° C. for 2 min, and 72° C. for 2 min for 40 cycles.

PCR products of approximately 280 bp were gel-purified, digested withEco R1, gel-purified again, and subcloned in the Bluescript vector(Stratagene, San Diego, Calif.). Bacterial colonies carrying individualsubclones were picked into 96 well microtiter plates, and multiplereplicas were prepared by plating the cells onto nitrocellulose. Thereplicate filters were hybridized to probes representing known membersof the family, and DNA was prepared from nonhybridizing colonies forsequence analysis.

The primer combination of SJL141 and SJL147, encoding the amino acidsequences GW (H/Q/N/K/D/E) (D/N)W(V/I/M) (V/I/M) (A/S)P (SEQ ID NO:9)and M(V/I/M/T/A)V(D/E)SC(G/A) C (SEQ ID NO:10), respectively, yieldedfour previously identified sequences (BMP-4, inhibin, βB, GDF-3 andGDF-5) and one novel sequence, which was designated GDF-8, among 110subclones analyzed.

Human GDF-8 was isolated using the primers: (SEQ ID NO:3) ACM13:5′-CGCGGATCCAGAGTCAAGGTGACAGACACAC-3′; and (SEQ ID NO:4) ACM14:5′-CGCGGATCCTCCTCATGAGCACCCACAGCGGTC-3′

PCR using these primers was carried out with one μg human genomic DNA at94° C. for 1 min, 58° C. for 2 min, and 72° C. for 2 min for 30 cycles.The PCR product was digested with Bam H1, gel-purified, and subcloned inthe Bluescript vector (Stratagene, San Francisco, Calif.).

EXAMPLE 2 Expression Pattern and Sequence of GDF-8

To determine the expression pattern of GDF-8, RNA samples prepared froma variety of adult tissues were screened by Northern analysis. RNAisolation and Northern analysis were carried out as described previously(Lee, S. J., Mol. Endocrinol., 4:1034, 1990) except that hybridizationwas carried out in 5×SSPE, 10% dextran sulfate, 50% formamide, 1% SDS,200 μg/ml salmon DNA, and 0.1% each of bovine serum albumin, ficoll, andpolyvinylpyrrolidone. Five micrograms of twice poly A-selected RNAprepared from each tissue (except for muscle, for which only 2 μg RNAwas used) were electrophoresed on formaldehyde gels, blotted, and probedwith GDF-8. As shown in FIG. 1, the GDF-8 probe detected a single mRNAspecies expressed at highest levels in muscle and at significantly lowerlevels in adipose tissue.

To obtain a larger segment of the GDF-8 gene, a mouse genomic librarywas screened with a probe derived from the GDF-8 PCR product. Thepartial sequence of a GDF-8 genomic clone is shown in FIG. 2 a. Thesequence contains an open reading frame correspond-ing to the predictedC-terminal region of the GDF-8 precursor protein. The predicted GDF-8sequence contains two potential proteolytic processing sites, which areboxed. Cleavage of the precursor at the second of these sites wouldgenerate a mature C terminal fragment 109 amino acids in length with apredicted molecular weight of 12,400. The partial sequence of humanGDF-8 is shown in FIG. 2 b. Assuming no PCR-induced errors during theisolation of the human clone, the human and mouse amino acid sequencesin this region are 100% identical.

The C-terminal region of GDF-8 following the putative proteolyticprocessing site shows significant homology to the known members of theTGF-β; superfamily (FIG. 3). FIG. 3 shows the alignment of theC-terminal sequences of GDF-8 with the corresponding regions of humanGDF-1 (Lee, Proc. Natl. Acad. Sci. USA, 88: 4250-4254, 1991), humanBMP-2 and 4 (Wozney, et al., Science, 242: 1528-1534, 1988), human Vgr-1(Celeste, et al. Proc. Natl. Acad. Sci. USA, 87: 9843-9847, 1990), humanOP-1 (Ozkaynak, et al., EMBO J, 9: 2085-2093, 1990), human BMP-5(Celeste, et al., Proc. Natl. Acad. Sci. USA, 87: 9843-9847, 1990),human BMP-3 (Wozney, et al., Science, 242: 1528-1534, 1988), human MiS(Cate, et al. Cell, 45: 685-698, 1986), human inhibin alpha, βA, and βB(Mason, et al., Biochem, Biophys. Res. Commun., 135: 957-964, 1986),human TGF-β1 (Derynck, et al., Nature, 316: 701-705, 1985), humanTGF-R2(deMartin, et al., ELMO J, 6: 3673-3677, 1987), and human TGF-β3 (tenDijke, et al., Proc. Natl. Acad. Sci. USA, 85: 4715-4719, 1988). Theconserved cysteine residues are boxed. Dashes denote gaps introduced inorder to maximize the alignment.

GDF-8 contains most of the residues that are highly conserved in otherfamily members, including the seven cysteine residues with theircharacteristic spacing. Like the TGF-βs and inhibin βs, GDF-8 alsocontains two additional cysteine residues. In the case of TGF-β2, thesetwo additional cysteine residues are known to form an intramoleculardisulfide bond (Daopin, et al., Science, 257:369, 1992; Schlunegger andGrutter, Nature, 358:430, 1992).

FIG. 4 shows the amino acid homologies among the different members ofthe TGF-β superfamily. Numbers represent percent amino acid identitiesbetween each pair calculated from the first conserved cysteine to the Cterminus. Boxes represent homologies among highly-related members withinparticular subgroups. In this region, GDF-8 is most homologous to Vgr-1(45% sequence identity).

EXAMPLE 3 Isolation of cDNA Clones Encoding Murine and Human GDF-8

In order to isolate full-length cDNA clones encoding murine and humanGDF-8, cDNA libraries were prepared in the lambda ZAP II vector(Stratagene) using RNA prepared from skeletal muscle. From 5 μg of twicepoly A-selected RNA prepared from murine and human muscle, cDNAlibraries consisting of 4.4 million and 1.9 million recombinant phage,respectively, were constructed according to the instructions provided byStratagene. These libraries were screened without amplification. Libraryscreening and characterization of cDNA inserts were carried out asdescribed previously (Lee, Mol. Endocrinol., 4: 1034-1040).

From 2.4×10⁶ recombinant phage screened from the murine muscle cDNAlibrary, greater than 280 positive phage were identified using a murineGDF-8 probe derived from a genomic clone, as described in Example 1. Theentire nucleotide sequence of the longest cDNA insert analyzed is shownin FIG. 5 a and SEQ ID NO:11. The 2676 base pair sequence contains asingle long open reading frame beginning with a methionine codon atnucleotide 104 and extending to a TGA stop codon at nucleotide 1232.Upstream of the putative initiating methionine codon is an in-frame stopcodon at nucleotide 23. The predicted pre-pro-GDF-8 protein is 376 aminoacids in length. The sequence contains a core of hydrophobic amino acidsat the N-terminus suggestive of a signal peptide for secretion (FIG. 6a), one potential N-glycosylation site at asparagine 72, a putative RXXRproteolytic cleavage site at amino acids 264-267, and a C-terminalregion showing significant homology to the known members of the TGF-βsuperfamily. Cleavage of the precursor protein at the putative RXXR sitewould generate a mature C-terminal GDF-8 fragment 109 amino acids inlength with a predicted molecular weight of approximately 12,400.

From 1.9×10⁶ recombinant phage screened from the human muscle cDNAlibrary, 4 positive phage were identified using a human GDF-8 probederived by polymerase chain reaction on human genomic DNA. The entirenucleotide sequence of the longest cDNA insert is shown in FIG. 5 b andSEQ ID NO:13. The 2743 base pair sequence contains a single long openreading frame beginning with a methionine codon at nucleotide 59 andextending to a TGA stop codon at nucleotide 1184. The predictedpre-pro-GDF-8 protein is 375 amino acids in length. The sequencecontains a core of hydrophobic amino acids at the N-terminus suggestiveof a signal peptide for secretion (FIG. 6 b), one potentialN-glycosylation site at asparagine 71, and a putative RX) (R proteolyticcleavage site at amino acids 263-266. FIG. 7 shows a comparison of thepredicted murine (top) and human (boKom) GDF-8 amino acid sequences.Numbers indicate amino acid position relative to the N-terminus.Identities between the two sequences are denoted by a vertical line.Murine and human GDF-8 are approximately 94% identical in the predictedpro-regions and 100% identical following the predicted RXXR cleavagesites.

EXAMPLE 4 Dimerization of GDF-8

To determine whether the processing signals in the GDF-8 sequence arefunctional and whether GDF-8 forms dimers like other members of theTGF-β superfamily, the GDF-8 cDNA was stably expressed in CHO cells. TheGDF-8 coding sequence was cloned into the pMSXND expression vector (Leeand Nathans, J. Biol. Chem., 263:3521, (1988) and transfected into CHOcells. Following G418 selection, the cells were selected in 0.2 μMmethotrexate, and conditioned medium from resistant cells wasconcentrated and electrophoresed on SDS gels. Conditioned medium wasprepared by Cell Trends, Inc. (Middletown, Md.). For preparation ofanti-GDF-8 serum, the C-terminal region of GDF-8 (amino acids 268 to376) was expressed in bacteria using the RSET vector (Invitrogen, SanDiego, Calif.), purified using a nickle chelate column, and injectedinto rabbits. All immunizations were carried out by Spring Valley Labs(Woodbine, Md.). Western analysis using [¹²⁵I] iodoprotein A was carriedout as described (Burnette, W. N., Anal. Biochem., 112:195, 1981).Western analysis of conditioned medium prepared from these cells usingan antiserum raised against a bacterially-expressed C-terminal fragmentof GDF-8 detected two protein species with apparent molecular weights ofapproximately 52K and 15K under reducing conditions, consistent withunprocessed and processed forms of GDF-8, respectively. No bands wereobtained either with preimmune serum or with conditioned medium from CHOcells transfected with an antisense construct. Under non-reducingconditions, the GDF-8 antiserum detected two predominant protein specieswith apparent molecular weights of approximately 101K and 25K,consistent with dimeric forms of unprocessed and processed GDF-8,respectively. Hence, like other TGF-β family members, GDF-8 appears tobe secreted and proteolytically processed, and the C-terminal regionappears to be capable of forming a disulfide-linked dimer.

EXAMPLE 5 Preparation of Antibodies Against GDF-8 and Expression ofGDF-8 in Mammalian Cells

In order to prepare antibodies against GDF-8, GDF-8 antigen wasexpressed as a fusion protein in bacteria. A portion of murine GDF-8cDNA spanning amino acids 268-376 (mature region) was inserted into thepRSET vector (Invitrogen) such that the GDF-8 coding sequence was placedin frame with the initiating methionine codon present in the vector; theresulting construct created an open reading frame encoding a fusionprotein with a molecular weight of approximately 16,600. The fusionconstruct was trans-formed into BL21 (DE3) (pLysS) cells, and expressionof the fusion protein was induced by treatment withisopropylthio-β-galactoside as described (Rosenberg, et al., Gene, 56:125-135). The fusion protein was then purified by metal chelatechromatography according to the instructions provided by Invitrogen. ACoomassie blue-stained gel of unpurified and purified fusion proteins isshown in FIG. 8.

The purified fusion protein was used to immunize both rabbits andchickens. Immization of rabbits was carried out by Spring Valley Labs(Sykesville, Md.), and immunization of chickens was carried out by HRP,Inc. (Denver, Pa.). Western analysis of sera both from immunized rabbitsand from immunized chickens demonstrated the presence of antibodiesdirected against the fusion protein.

To express GDF-8 in mammalian cells, the murine GDF-8 cDNA sequence fromnucleotides 48-1303 was cloned in both orientations downstream of themetallothionein I promoter in the pMSXND expression vector; this vectorcontains processing signals derived from SV40, a dihydrofolate reductasegene, and a gene conferring resistance to the antibiotic G418 (Lee andNathans, J. Biol. Chem., 263: 3521-3527). The resulting constructs weretransfected into Chinese hamster ovary cells, and stable tranfectantswere selected in the presence of G418. Two milliliters of conditionedmedia prepared from the G418-resistant cells were dialyzed, lyophilized,electrophoresed under denaturing, reducing conditions, transferred tonitrocellulose, and incubated with anti-GDF-8 antibodies (describedabove) and [¹²⁵I] iodoproteinA.

As shown in FIG. 9, the rabbit GDF-8 antibodies (at a 1:500 dilution)detected a protein of approximately the predicted molecular weight forthe mature C-terminal fragment of GDF-8 in the conditioned media ofcells transfected with a construct in which GDF-8 had been cloned in thecorrect (sense) orientation with respect to the metallothionein promoter(lane 2); this band was not detected in a similar sample prepared fromcells transfected with a control antisense construct (lane 1). Similarresults were obtained using antibodies prepared in chickens. Hence,GDF-8 is secreted and proteolytically processed by these transfectedmammalian cells.

EXAMPLE 6 Expression Pattern of GDF-8

To determine the pattern of GDF-8, 5 μg of twice poly A-selected RNAprepared from a variety of murine tissue sources were subjected toNorthern analysis. As shown in FIG. 10 a (and as shown previously inExample 2), the GDF-8 probe detected a single mRNA species presentalmost exclusively in skeletal muscle among a large number of adulttissues surveyed. On longer exposures of the same blot, significantlylower but detectable levels of GDF-8 mRNA were seen in fat, brain,thymus, heart, and lung. Hence, these results confirm the high degree ofspecificity of GDF-8 expression in skeletal muscle. GDF-8 mRNA was alsodetected in mouse embryos at both gestational ages (day 12.5 and day18.5 post-coital) examined but not in placentas at various stages ofdevelopment (FIG. 10 b).

To further analyze the expression pattern of GDF-8, in situhybridization was performed on mouse embryos isolated at various stagesof development. For all in situ hybridization experiments, probescorresponding to the C-terminal region of GDF-8 were excluded in orderto avoid possible cross-reactivity with other members of thesuperfamily. Whole mount in situ hybridization analysis was carried outas described (Wilkinson, D. G., In Situ Hybridization, A PracticalApproach, pp. 75-83, IRL. Press, Oxford, 1992) except that blocking andantibody incubation steps were carried out as in Knecht et al. (Knecht,et al., Development, 121:1927, 1955). Alkaline phosphatase reactionswere carried out for 3 hours for day 10.5 embryos and overnight for day9.5 embryos. Hybridization was carried out using digoxigenin-labelledprobes spanning nucleotides 8-811 and 1298-2676, which correspond to thepro-region and 3′ untranslated regions, respectively. In situhybridization to sections was carried out as described (Wilkinson, etal., Cell, 50:79, 1987) using ³⁵S-labelled probes ranging fromapproximately 100-650 bases in length and spanning nucleotides 8-793 and1566-2595. Following hybridization and washing, slides were dipped inNTB-3 photographic emulsion, exposed for 16-19 days, developed andstained with either hematoxylin and eosin or toluidine blue. RNAisolation, poly A selection, and Northern analysis were carried out asdescribed previously (McPherron and Lee, J. Biol. Chem., 268:3444,1993).

At all stages examined, the expression of GDF-8 mRNA appeared to berestricted to developing skeletal muscle. At early stages, GDF-8expression was restricted to developing somites. By whole mount in situhybridization analysis, GDF-8 mRNA could first be detected as early asday 9.5 post coitum in approximately one-third of the somites. At thisstage of development, hybridization appeared to be restricted to themost mature (9 out of 21 in this example), rostral somites. By day 10.5p.c., GDF-8 expression was clearly evident in almost every somite (28out of 33 in this example shown). Based on in situ hybridizationanalysis of sections prepared from day 10.5 p.c. embryos, the expressionof GDF-8 in somites appeared to be localized to the myotome compartment.At later stages of development, GDF-8 expression was detected in a widerange of developing muscles.

GDF-8 continues to be expressed in adult animals as well. By Northernanalysis, GDF-8 mRNA expression was seen almost exclusively in skeletalmuscle among the different adult tissues examined. A significantly lowerthough clearly detectable signal was also seen in adipose tissue. Basedon Northern analysis of RNA prepared from a large number of differentadult skeletal muscles, GDF-8 expression appeared to be widespreadalthough the expression levels varied among individual muscles.

EXAMPLE 7 Chromosomal Localization of GDF-8

In order to map the chromosomal location of GDF-8, DNA samples fromhuman/rodent somatic cell hybrids (Drwinga, et al., Genomics, 16:311-413, 1993; Dubois and Naylor, Genomics, 16: 315-319, 1993) wereanalyzed by polymerase chain reaction followed by Southern blotting.Polymerase chain reaction was carried out using primer #83,5′-CGCGGATCCGTGGATCTAAATGAGAACAGTGAGC-3′ (SEQ ID NO: 15) and primer #84,5′-CGCGAATTCTCAGGTAATGATTGTTTCCGTTGTAGCG-3′ (SEQ ID NO:16) for 40 cyclesat 94° C. for 2 minutes, 60° C. for 1 minute, and 72° C. for 2 minutes.These primers correspond to nucleotides 119 to 143 (flanked by a Bam H1recognition sequence), and nucleotides 394 to 418 (flanked by an Eco R1recognition sequence), respectively, in the human GDF-8 cDNA sequence.PCR products were electrophoresed on agarose gels, blotted, and probedwith oligonucleotide #100, 5′-ACACTAAATCTTCAAGAATA-3′ (SEQ ID NO:17),which corresponds to a sequence internal to the region flanked by primer#83 and #84. Filters were hybridized in 6×SSC, 1× Denhardt's solution,100 T g/ml yeast transfer RNA, and 0.05% sodium pyrophosphate at 50° C.

As shown in FIG. 11, the human-specific probe detected a band of thepredicted size (approximately 320 base pairs) in the positive controlsample (total human genomic DNA) and in a single DNA sample from thehuman/rodent hybrid panel. This positive signal corresponds to humanchromosome 2. The human chromosome contained in each of the hybrid celllines is identified at the top of each of the first 24 lanes (1-22, X,and Y). In the lanes designated M, CHO, and H, the starting DNA templatewas total genomic DNA from mouse, hamster, and human sources,respectively. In the lane marked B1, no template DNA was used. Numbersat left indicate the mobilities of DNA standards. These data show thatthe human GDF-8 gene is located on chromosome 2.

EXAMPLE 8 GDF-8 Transgenic Knockout Mice

The GDF-8, we disrupted the GDF-8 gene was disrupted by homologoustargeting in embryonic stem cells. To ensure that the resulting micewould be null for GDF-8 function, the entire mature C-terminal regionwas deleted and replaced by a neo cassette (FIG. 12 a). A murine 129SV/J genomic library was prepared in lambda FIX II according to theinstructions provided by Stratagene (La Jolla, Calif.). The structure ofthe GDF-8 gene was deduced from restriction mapping and partialsequencing of phage clones isolated from this library. Vectors forpreparing the targeting construct were kindly provided by Philip Sorianoand Kirk Thomas University. R1 ES cells were transfected with thetargeting construct, selected with gancyclovir (2 μM) and G418 (250μg/ml), and analyzed by Southern analysis. Homologously targeted cloneswere injected into C57BL/6 blastocysts and transferred intopseudopregnant females. Germline transmission of the targeted allele wasobtained in a total of 9 male chimeras from 5 independently-derived ESclones. Genomic Southern blots were hybridized at 42° C. as describedabove and. washed in 0.2×SSC, 0.1% SDS at 42° C.

For whole leg analysis, legs of 14 week old mice were skinned, treatedwith 0.2 M EDTA in PBS at 4° C. for 4 weeks followed by 0.5 M sucrose inPBS at 4° C. For fiber number and size analysis, samples were directlymounted and frozen in isopentane as described (Brumback and Leech, ColorAtlas of Muscle Histochemistry, pp. 9-33, PSG Publishing Company,Littleton, Mass., 1984). Ten to 30 μm sections were prepared using acryostat and stained with hematoxylin and eosin. Muscle fiber numberswere determined from sections taken from the widest part of the tibialiscranialis muscle. Muscle fiber sizes were measured from photographs ofsections of tibialis cranialis and gastrocnemius muscles. Fiber typeanalysis was carried out using the mysosin ATPase assay afterpretreatment at pH 4.35 as described (Cumming, et al., Color Atlas ofMuscle Pathology, pp. 184-185, 1994) and by immunohistochemistry usingan antibody directed against type I myosin (MY32, Sigma) and theVectastain method (Vector Labs); in the immunohistochemical experiments,no staining was seen when the primary antibodies were left out.Carcasses were prepared from shaved mice by removing the all of theinternal organs and associated fat and connective tissue. Fat content ofcarcasses from 4 month old males was determined as described (Leshner,et al., Physiol. Behavior, 9:281, 1972).

For protein and DNA analysis, tissue was homogenized in 150 mM NaCl, 100mM EDTA. Protein concentrations were determined using the Biorad proteinassay. DNA was isolated by adding SDS to 1%, treating with 1 mg/mlproteinase K overnight at 55° C., extracting 3 times with phenol andtwice with chloroform, and precipitating with ammonium acetate and EtOH.DNA was digested with 2 mg/ml RNase for 1 hour at 37° C., and followingproteinase K digestion and phenol and chloroform extractions, the DNAwas precipitated twice with ammonium acetate and EtOH.

Homologous targeting of the GDF-8 gene was seen in 13/131gancyclovir/G418 doubly-resistant ES cell clones. Following injection ofthese targeted clones into blastocysts, we obtained chimeras from 5independently-derived ES clones that produced heterozygous pups whencrossed to C57BL/6 females (FIG. 12 b). Genotypic analysis of 678offspring derived from crosses of F1 heterozygotes showed 170+/+(25%),380+/− (56%), and 128−/− (19%). Although the ratio of genotypes wasclose to the expected ratio of 1:2:1, the smaller than expected numberof homozygous mutants appeared to be statistically significant(p<0.001).

Homozygous mutants were viable and fertile when crossed to C57BL/6 miceand to each other. Homozygous mutant animals, however, wereapproximately 30% larger than their heterozygous and wild typelittermates (Table 1). The difference between mutant and wild type bodyweights appeared to be relatively constant irrespective of age and sexin adult animals. Adult mutants also displayed an abnormal body shape,with pronounced shoulders and hips. When the skin was removed fromanimals that had been sacrificed, it was apparent that the muscles ofthe mutants were much larger than those of wild type animals. Theincrease in skeletal muscle mass appeared to be widespread throughoutthe body. Individual muscles isolated from homozygous mutant animalsweighed approximately 2-3 times more than those isolated from wild typelittermates (Table 2). Although the magnitude of the weight increaseappeared to roughly correlate with the level of GDF-8 expression in themuscles examined. To determine whether the increased muscle mass couldaccount for the entire difference in total body weights between wildtype and mutant animals or whether many tissues were generally larger inthe mutants, we compared the total body weights to carcass weights. Asshown in Table 3, the difference in carcass weights between wild typeand mutant animals was comparable to the difference in total bodyweights. Moreover, because the fat content of mutant and wild typeanimals was similar, these data are consistent with all of the totalbody weight difference resulting from an increase in skeletal musclemass, although we have not formally ruled out the possibility thatdifferences in bone mass might also contribute to the differences intotal body mass.

To determine whether the increase in skeletal muscle mass resulted fromhyperplasia or from hypertrophy, histologic analysis of severaldifferent muscle groups was performed. The mutant muscle appearedgrossly normal. No excess connective tissue or fat was seen nor werethere any obvious signs of degeneration, such as widely varying fibersizes (see below) or centrally-placed nuclei. Quantitation of the numberof muscle fibers showed that at the widest portion of the tibialiscranialis muscle, the total cell number was 86% higher in mutant animalscompared to wild type littermates [mutant 5470+/−121 (n=3), wildtype=2936+/−288 (n=3); p<0.01]. Consistent with this result was thefinding that the amount of DNA extracted from mutant muscle was roughly50% higher than from wild type muscle [mutant=350 μg (n=4), wildtype=233 μg (n=3) from pooled gastrocnemius, plantaris, triceps brachii,tibialis cranialis, and pectoralis muscles; p=0.05]. Hence, a large partof the increase in skeletal muscle mass resulted from muscle cellhyperplasia. However, muscle fiber hypertrophy also appeared tocontribute to the overall increase in muscle mass. As shown in FIG. 13,the mean fiber diameter of the tibialis cranialis muscle andgastrocnemius muscle was 7% and 22% larger, respectively, in mutantanimals compared to wild type littermates, suggesting that thecross-sectional area of the fibers was increased by approximately 14%and 49%, respectively. Notably, although the mean fiber diameter waslarger in the mutants, the standard deviation in fiber sizes was similarbetween mutant and wild type muscle, consistent with the absence ofmuscle degeneration in mutant animals. The increase in fiber size wasalso consistent with the finding that the protein to DNA ratio (w/w) wasslightly increased in mutant compared to wild type muscle[mutant=871+/−111 (n=4), wild type=624+/−85 (n=3); p<0.05].

Finally, fiber type analysis of various muscles was carried out todetermine whether the number of both type I (slow) and type II (fast)fibers was increased in the mutant animals. In most of the musclesexamined, including the tibialis cranialis muscle, the vast majority ofmuscle fibers were type II in both mutant and wild type animals. Hence,based on the cell counts discussed above, the absolute number of type IIfibers were increased in the tibialis cranialis muscle. In the soleusmuscle, where the number of type I fibers was sufficiently high that wecould attempt to quantitate the ratio of fiber types could bequantiated, the percent of type I fibers was decreased by approximately33% in mutant compared to wild type muscle [wild type=39.2+/−8.1 (n=3),mutant 26.4+/−9.3 (n=4)]; however, the variability in this ratio forboth wild type and mutant animals was too high to support any firmconclusions regarding the relative number of fiber types.

EXAMPLE 9 Isolation of Rat and Chicken GDF-8

In order to isolate rat and chicken GDF-8 cDNA clones, skeletal musclecDNA libraries prepared from these species were obtained from Stratageneand screened with a murine GDF-8 probe. Library screening was carriedout as described previously (Lee, Mol. Endocranial., 4: 1034-1040)except that final washes were carried out in 2×SSC at 65° C. Partialsequence analysis of hybridizing clones revealed the presence of openreading frames highly related to murine and human GDF-8. Partialsequences of rat and chicken GDF-8 are shown in FIGS. 2 c and 2 d,respectively, and an alignment of the predicated rat and chicken GDF-8amino acid sequences with those of murine and human GDF-8 are shown inFIG. 3 b. All four sequences contain an RSRR sequence that is likely torepresent the proteolytic processing site. Following this RSRR sequence,the sequences contain a C-terminal region that is 100% conserved amongall four species. The absolute conservation of the C-terminal regionbetween species as evolutionarily far apart as humans and chickenssuggests that this region will be highly conserved in many other speciesas well.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A method for increasing the muscle mass in an animal comprisingadministering to the animal an antibody, or functional fragment thereof,which binds to GDF-8 polypeptide, thereby increasing muscle mass in theanimal. 2-19. (canceled)
 20. Substantially pure growth differentiationfactor-8 (GDF-8) having the amino acid sequence as set forth in SEQ IDNO:21.
 21. An isolated polynucleotide encoding the GDF-8 polypeptidehaving the amino acid sequence as set forth in SEQ ID NO:21.
 22. Anisolated polynucleotide selected from the group consisting of: a) SEQ IDNO:20; b) SEQ ID NO:20, wherein T can also be U; c) nucleic sequencescomplementary to SEQ ID NO:20; and d) fragments of a) b), or c) that areat least 15 bases in length and that will selectively hybridize togenomic DNA which encodes the GDF-8 protein of SEQ ID NO:21.
 23. Thepolynucleotide of claim 21, wherein the polynucleotide is isolated froman avian cell.
 24. The polynucleotide of claim 23, wherein the aviancell is a chicken or turkey cell.
 25. An expression vector including thepolynucleotide of claim
 21. 26. The vector of claim 25, wherein thevector is a plasmid.
 27. The vector of claim 25, wherein the vector is aviral vector.
 28. A host cell containing the vector of claim
 25. 29. Thehost cell of claim 28, wherein the cell is prokaryotic.
 30. The hostcell of claim 28, wherein the cell is eukaryotic.